Vascular endothelial growth factors (VEGFs) are considered as key growth factors inducing angiogenesis and lymphangiogenesis during embryogenesis, as well as maintaining vasculature during adulthood. Their abnormal expression is also found on several pathological conditions such as cancer and retinopathies. VEGF-A belongs to the larger family of related growth factors including VEGF-B, -C, -D and placental growth factor PlGF as well as Orf virus derived VEGF-E proteins and multiple homologues from snake venoms. Endogenous VEGF protein family members in humans exist as several isoforms either as a result of alternative splicing of the mRNAs or due to proteolytic processing. The angiogenic effects of these variants vary considerably due to their differing specificities and affinities to three main VEGF receptors, co-receptors such as neuropilins, heparan sulphate proteoglycans and other components of the extracellular matrix.
VEGFR-2 is the most important receptor regulating angiogenesis and it is mainly expressed on endothelial cells. Mammalian VEGFR-2 ligands include VEGF-A, VEGF-C and VEGF-D. In addition to VEGFR-2 VEGF-C and -D are ligands of VEGFR-3 which is the receptor mediating lymphangiogenesis and partakes therefore in the formation of lymphatic vasculature. VEGF-A binds also to VEGFR-1 which functions during embryogenesis mainly as a non-signalling decoy receptor. In adult organism this receptor is known to mediate migration of inflammatory cells such as macrophages and monocytes but its role in angiogenesis is still controversial.
Due to their importance as angiogenic regulators, the VEGF family members have been suggested as potential therapeutics in order to adjust the angiogenic processes in different pathological conditions (Ylä-Herttuala 2003). In vivo studies have been done to induce angiogenesis by introducing VEGFs to tissues either directly as recombinant proteins or using gene therapy vectors (Markkanen 2005). The findings from several studies have shown that VEGF family members have strong angiogenic activity in vivo and they are potentially useful therapeutics for conditions like lower limb ischemia and coronary artery disease. Out of these factors, the mature form of VEGF-D (VEGF-DΔNΔC, see below) and VEGF-A have been found to be the most promising to induce therapeutic angiogenesis.
VEGFs share structural similarity with platelet-derived growth factors (PDGFs) and together they are classified as VEGF/PDGF family, which belongs to bigger cysteine knot growth factor superfamily. Family members share a cysteine knot motif which is found in many extracellular proteins and is conserved among numerous species. Characteristic to cysteine knot proteins is that they contain a conserved structure of antiparallel β-sheets connected by three disulfide bonds. Typically cysteine knot growth factors form dimers which in the case of VEGF/PDGF family are often linked by intersubunit disulfide bonds.
VEGF receptors belong to receptor protein tyrosine kinases which are activated by dimerization. For VEGFR activation, dimerization of the ligand is indispensable. One VEGF-A dimer binds from its both poles to two separate receptor monomers, inducing receptor dimerization and consequently intracellular tyrosine kinase activity. Based on the several experimentally solved 3D structures of VEGF family members either free or as a complex with VEGF receptor, they all have closely similar tertiary structures and so probably induce receptor activation by similar mechanisms.
In the VEGF family, VEGF-C and VEGF-D can be subdivided into their own subfamily, which is reflected by their higher primary sequence structure similarity as compared to other VEGFs. There are several characterising features including: 1) they are the only VEGFs that bind to VEGFR-3, the lymphangiogenesis mediating receptor; 2) by contrast to VEGF-A, -B and PLGF, VEGF-C and VEGF-D are expressed as long preproteins. These forms have poor receptor-binding affinities and, in order to be converted to more active growth factors, VEGF-C and VEGF-D are proteolytically processed both from their N-terminal and C-terminal ends; 3) in contrast to other members of the family, the mature proteolytically processed form of VEGF-D, VEGF-DΔNΔC, has been found to exist mainly as a non-covalently bound dimer or monomer and only in small degree as a covalently bound disulfide bond-linked dimer. These studies have also shown that the monomeric fraction of VEGF-DΔNΔC is also only very weakly active when compared to the dimeric fraction. The mainly non-covalent nature of the dimers is somewhat surprising, since the cysteine residues that form the intersubunit linkage in other VEGF family growth factors are conserved in the VEGF-D protein.
The cysteines of VEGF-A involved in cysteine knot structure have been mutagenized in previous studies to investigate their importance for the structure and function of the protein. The intersubunit disulfide bonds have been found to be necessary for its biological function, as VEGF-A where these cysteines have been mutated to alanines has lost its biological activity. A VEGF-C mutant where one of the conserved cysteines (Cys156) has been converted to serine has completely lost its VEGFR-2 activation ability, but is still able to activate VEGFR-3. Both mature forms of VEGF-C and VEGF-D also contain an unpaired cysteine residue located close to the proposed intersubunit disulfide bonds forming cysteine residues. The pvf-1 gene from C. elegans has been recently shown to code for a VEGF/PDGF homolog that activates human VEGFR-1 and -2 and is also only partially covalently bound dimer. This protein also has a unpaired cysteine on the dimer interface, like VEGF-C and VEGF-D.